Summary of work: The insulin receptor is a homodimer consisting of two alpha- and two beta-subunits linked together by disulfide bonds. Insulin binding to the alpha-subunit triggers the intrinsic tyrosine kinase function of the cytoplasmic domain of the receptor beta-subunit. Activation of this receptor by insulin plays an important role in mediating diverse physiological processes, including modulation of glucose homeostasis and gene expression. Earlier findings have found that the functional activity of the insulin receptor can be modulated by redox regulation in the cellular environment. For example, addition of antioxidant such as N-acetyl-L-cysteine (NAC) to cells has been associated with a reduction in insulin receptor catalytic function, whereas cell treatment with hydrogen peroxide promotes insulinomimetic effects. We postulated that reduced glutathione (GSH), which constitutes the major source of plasma nonprotein thiols, and NAC may play a role in altering thiol reactivity of the insulin receptor and thus reduce insulin responsiveness. In a recent published study, we provided the first evidence that the insulin receptor alpha-subunit contains a select group of disulfides whose redox status can be rapidly and reversibly altered by the reducing agents GSH and NAC. While having little impact on insulin binding, GSH markedly attenuated insulin signal transduction. The oligomeric structure of the insulin receptor was not affected by neither of these compounds. We plan to identify the reactive receptor thiol group(s). Such knowledge will allow mutagenic structure/function analysis of this select group of insulin receptor disulfides.